The EXCOR Ventricular Assist Device (VAD) system (Berlin Heart GmbH, Berlin, Germany) is used for mechanical circulatory support in adult and pediatric patients with advanced heart failure as bridging to transplantation or to myocardial recovery.1–3 Pulsatile blood flow is generated by an external pump, which is attached via inflow and outflow cannulas to the native heart and the great arteries of the patient. The EXCOR pump consists of two chambers; one for blood and one for air, separated by a flexible triple-layer membrane. The housing and membrane are made from biocompatible, semirigid polyurethane.
A critical factor of VAD systems is the exposure of the circulating blood to foreign surfaces, which can lead to thrombus formation, embolization, and device failure as a result of activation of platelets and of the coagulation system.4 To reduce problems related to the exposure of blood to foreign materials, Berlin Heart GmbH in 1994 started to integrate a hemocompatible surface coating (CARMEDA BioActive Surface [CBAS, Carmeda, AB, Upplands, Väsby, Sweden ]) in the EXCOR manufacturing process. The CBAS Heparin Surface, which is applied to all blood-contacting surfaces of the device, consists of a polymeric base matrix to which heparin is bound in a way that preserves the anticoagulant activity by using end-point attachment.5 The anticoagulant activity of the end-point-attached heparin, referred to as bioactivity,6 is measured as the capacity to bind antithrombin, a natural coagulation inhibitor in blood and the key protein for the heparin-mediated control of blood coagulation. Clinical applications of the CBAS Heparin Surface include short-term procedures such as cardiopulmonary bypass,7 long-term procedures such as VADs,8 and permanent implants such as vascular grafts and stent grafts.9 The effectiveness in reducing surface-induced thrombosis and even inflammation has also been demonstrated in a number of in vitro studies.10,11 For EXCOR, the CBAS Heparin Surface has been shown to reduce the frequency and thickness of thrombotic deposits, thereby likely reducing complications related to thromboembolism.8
Apart from an anecdotal report showing that the CBAS Heparin Surface of an EXCOR pump remained intact after 855 days in a patient,12 no studies on the durability of the CBAS Heparin Surface in EXCOR pumps during long-term clinical use have been published previously. In the current study, the CBAS Heparin Surface on samples derived from retrieved EXCOR devices was analyzed for the surface density and bioactivity of the end-point-attached heparin.
EXCOR pumps (n = 15) of sizes 50, 60, and 80 ml retrieved after clinical use were included in the study.
Collection and Shipment of Devices
Pumps were collected immediately after clinical use. Blood residues and visible debris were removed by rinsing with physiologic saline (0.15 M NaCl). The devices were then filled with physiologic saline containing 10 mM ethylenediaminetetraacetic acid and 0.02% sodium azide to prevent microbial growth and shipped to Carmeda for analysis.
Upon arrival, the EXCOR pumps were washed with physiologic saline, after which surface-adsorbed proteins were removed by incubation with 0.5% sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS) overnight. The devices were then further cleaned by incubation with 1% SDS in PBS followed by 2 M NaCl, both overnight. After each cleaning step, the units were rinsed with physiologic saline.
After cleaning, 12 disk-shaped samples (diameter 14 mm) were punched out from each EXCOR pump for analysis: six from the semirigid housing wall and six from the flexible membrane. The analyzed EXCOR pumps were equipped with titanium tilting disks that did not allow punching of samples. Three samples of each origin (chamber and membrane) were assayed for surface heparin density and the remaining three for surface heparin bioactivity.
Heparin Density Assay
Quantitative analysis of surface-bound heparin was performed by complete degradation followed by colorimetric determination of the reaction products released into solution. Degradation was achieved by reacting the heparin surface with an excess of sodium nitrite under acidic conditions. The degradation products, mainly disaccharides, were quantified colorimetrically in a reaction with 3-methyl-2-bezotiazolinon hydrazone hydrochloride, essentially as described by Smith and Gilkerson.13
Heparin Bioactivity Assay
Heparin bioactivity was measured as the capacity of the surface-bound heparin to bind antithrombin.6 Washed samples were incubated with an excess antithrombin in solution to saturate all available antithrombin-binding sites of the heparin surface. Nonspecifically adsorbed antithrombin was rinsed away using a salt solution. Subsequently, antithrombin specifically bound to the surface-bound heparin was released by incubating with a solution of heparin at high concentration. Finally, the antithrombin released from the heparin surface was measured in a thrombin inhibition assay, based on a chromogenic thrombin substrate.
Mean with standard deviation and range data were calculated for all variables. Heparin density and bioactivity results versus implant duration were fitted to linear curves using least square regression. The hypothesis that there is no relationship between the tested parameter and the implant duration was tested using analysis of variance (α = 0.05). Comparison between groups was performed using Student’s t-test (α = 0.05). All statistical evaluations were performed using JMP 10 (SAS Institute Inc., Cary, NC).
Surface Heparin Bioactivity and Density of Collected Pumps
A total of 15 EXCOR pumps were retrieved after clinical use at two different clinics. Seven pumps were retrieved because of heart transplantation, and six pumps were retrieved from deceased patients. One pump was retrieved because of suspected thrombus formation. One device was excluded from the study and returned to Berlin Heart for technical analysis.
The duration of ventricular assistance ranged from 15 to 461 days, with a mean of 178 ± 160 days and a median of 114 days. Finished product EXCOR pumps had a mean heparin activity of 30.4 ± 6.6 pmol/cm2 (range, 19–41 pmol/cm2). Mean heparin bioactivity of all analyzed samples was 14 ± 5 ρmol/cm2 (range, 7–27 ρmol/cm2) and mean heparin density 3.1 ± 0.6 μg/cm2 (range: 2.2–4.8 μg/cm2). For details, see Table 1 and Figure 1.
Housing Versus Membrane
In Figure 2, the mean analytical results for the housing and membrane samples from each pump have been plotted as groups to determine whether the CBAS Heparin Surface differs between the semirigid housing wall and the flexing membrane. The mean heparin bioactivity of housing and membrane samples was 13 ± 5 ρmol/cm2 (range, 7–25 ρmol/cm2) and 14 ± 6 ρmol/cm2 (range, 5–28 ρmol/cm2), respectively (Figure 2A). The mean heparin density of housing and membrane samples was similar for both 3.1 ± 0.8 μg/cm2 (range, 1.1–4.4 μg/cm2versus 1.8–5.2 μg/cm2; Figure 2B). There were, however, no significant differences between housing and membrane, neither for heparin density (p = 0.95) nor for bioactivity (p = 0.78).
Effect of Time
In Figure 3, the mean heparin bioactivity (A) and density (B) results of each pump have been plotted versus the duration of ventricular assist. As seen from the linear trend lines included in the figures, there was no marked decline over time, indicating that the CBAS Heparin Surface remained stable.
In this study, the durability of the CBAS Heparin Surface of EXCOR VAD pumps, measured as heparin density and bioactivity, was evaluated. Analysis was performed on samples derived from EXCOR pumps retrieved after long-term clinical use. The results show that the CBAS Heparin Surface remained stable after implantation, independent of duration of clinical use.
A drop in bioactivity compared with final product is not unexpected for a biologically active substance. However, all tested EXCOR pumps expressed heparin bioactivities of 7 ρmol/cm2 or higher, which previously has been shown to be sufficient to maintain thromboresistance.14 In that study, bioactivity was measured before blood contact. The amount of immobilized heparin on the surface remained largely unchanged over time, as did the capacity to interact with antithrombin, showing that essentially no deterioration had occurred even after 461 days of clinical use. The stability of the CBAS Heparin Surface appeared similar for housing and membrane, which is remarkable in view of the challenge to the coating adherence the membrane flexing with every pump cycle must represent. It should be pointed out that the measured levels of heparin bioactivity most likely are affected by the thorough cleaning of the samples before analysis, exposing areas previously covered by proteins after extensive contact with blood during clinical use. Hence, the reported values might be higher than what would have been the case without cleaning. In future studies, it is also of interest to study the resulting bioactivity measured after a saline rinse only. Besides heparin bioactivity after protein exposure, there are also other parameters of interest to identify long-term effects. Gore et al.15 have demonstrated that the composition of adsorbed proteins differs compared with an uncoated surface.
The results are in line with the study by Kaufmann et al.8 on EXCOR devices analyzed after up to 190 days of clinical use. These authors showed that the replacement rate of pumps with the CBAS Heparin Surface was significantly reduced compared with uncoated pumps, because of decreased thrombus deposition and thromboembolic complications.8 There are also in vivo data up to 12 weeks available on the stability of the CBAS Heparin Surface on expanded polytetrafluoroethylene (ePTFE) vascular grafts reported from a canine implant model.16 Moreover, a randomized clinical study has demonstrated significantly improved patency rates for ePTFE grafts with the CBAS Heparin Surface compared with grafts without.17
In conclusion, this study shows that, after implantation of the EXCOR pumps, the CBAS Heparin Surface remains structurally and functionally intact on the device surface during clinical use, from the time of the first explant up to periods exceeding 1 year. To the best of our knowledge, similar data have not been published for other biocompatible coatings on medical devices retrieved after extensive clinical use. This study supports the mid-term and long-term clinical use of the EXCOR pump as a bridge-to-heart transplantation or to myocardial recovery for at least 1 year.
The authors acknowledge Mrs. Gunilla Sundin for the analysis of heparin bioactivity and heparin density. Dr. Johan Riesenfeld is acknowledged for reviewing the manuscript. The authors also acknowledge Mrs. Kersten Brandes for study initiation.
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